Lyme borreliosis is the most prevalent tick-borne disease in the United States as well as one of the most important tick-borne infectious diseases worldwide. It is endemic in much of the northeastern United States, Minnesota, Wis., and parts of the Pacific Northwest. The bacterial spirochete Borrelia burgdorferi is the causative agent for Lyme disease. Infection with B. burgdorferi produces local and systemic manifestations. Local symptoms that appear early after infection include a skin lesion at the site of the tick bite, termed erythema chronicum migrans (ECM), as well as fever and flu-like illness. Weeks to months after infection, systemic manifestations that include rheumatic, cardiac and neurological symptoms appear. The early local phase of B. burgdorferi infection is easily treatable with antibiotics. However, the later systemic phases have proved to be more refractory to antibiotics.
Much attention has been given to investigation of the immune response to B. burgdorferi infection in animal and human model systems, and how the initial phagocytic and subsequent humoral and cellular responses affect the pathogenesis of the disease. The success of initial phagocytic interactions with invading spirochetes quite probably influences the progression of disease, and thus has been the subject of much investigation.
There are numerous observations suggesting a cell-based response to infection. Several studies have demonstrated that neutrophils and monocytes bind and phagocytose both opsonised and non-opsonised B. burgdorferi of high and low passage strains. See, e.g. Peterson et al., Infect. Immun. (1984) 46:608-611. Opsonization of the spirochetes with specific immune serum greatly enhanced phagocytosis; this effect appears to be dependent on the availability of polymorphonuclear Fc receptors, and independent of heat dependent serum factors. Treatment of the spirochetes with normal serum [Benach et al., J. Infect. Dis. (1984) 150:497-507] or normal whole blood [Banfi et al., J. Applied Bact. (1989) 67:37-45] (non-specific opsonization) resulted in a phagocytosis rate intermediate between non-opsonized and immune serum opsonized treatments. Phagocytic cells are observed in the infiltrates of experimentally induced skin lesions in animal models and humans, as reported by Benach et al., Yale J. Biol. Med. (1984) 57:599-605. It is speculated that the cells participating in immediate responses may differ from those circulating at later times in the infective cycle. In addition, it has been shown that while natural killer (NK) cells numerically increase shortly after infection, the NK cell activity of infected individuals is depressed. This may be related to early expression of suppressor cell activity. Cellular response begins early and prior to a measurable humoral response.
For optimum effectiveness, antibiotic treatment of Lyme borreliosis should be administered early in the course of the disease, when spirochetemia and the polymorphonuclear leukocyte (PMN) phagocytic responses are prominent. Early, appropriate treatment aborts progression to more severe cardiac neurological and arthritic manifestations in the majority of cases. In addition to maximizing the cure rate and minimizing patient suffering, early treatment is more cost effective than less successful treatments for later arthritic and neurological manifestations of Lyme disease, which may include extended periods of intravenous antibiotic therapy. Thus, early diagnosis of Lyme disease is essential.
Lyme disease is currently difficult to definitively diagnose. The early symptoms are often non-specific. Further, although B. burgdorferi-infected Ixodid ticks are the predominant source of infection, some patients have no history of a tick bite and 25-30% of the patients do not develop ECM. A screening test which reliably detects B. burgdorferi infection early in the course of the disease would allow early diagnosis and treatment, and eliminate the need for the physician to treat patients based on clinical suspicion alone.
Currently available diagnostic tests, however, are inadequate. Although B. burgdorferi spirochetes have been demonstrated in the blood, urine, cerebrospinal fluid (CSF) and synovial fluid of infected animals and humans, their numbers are too few, and their presence too variable, to allow reliable diagnosis by traditional dark field examination. Culture of these organisms from body fluids is difficult due to the low number of spirochetes in samples and the slow growth rate of wild strain B. burgdorferi in culture; the process can take up to five weeks. Bacterial contamination of samples is another common difficulty, as the rich Barbour-Stoenner-Kelly (BSK) media needed to support B. burgdorferi growth will also encourage growth of contaminants, and the few antibiotics that can be used to decrease contamination also slow B. burgdorferi growth considerably.
The most commonly used diagnostic tests for Lyme disease, Enzyme Linked Immunosorbent Assay (ELISA) and Immune Fluorescent Antibody (IFA) techniques, measure antibodies to B. burgdorferi. These tests are often inaccurate, for several reasons. Clinical symptoms of Lyme disease often develop prior to the development of measurable antibody response. It may take as long as three to six weeks after infection for measurable IgM to develop, and months for IgG to reach measurable levels, while clinical signs may occur within two to twenty days following infection. In addition, certain individuals may not mount a measurable antibody response, development of antibody titer may be muted by early antibiotic treatment, and the sensitivity of antibody tests, especially IFA, varies widely among laboratories. The use of different B. burgdorferi strains as antigens leads to varied test results in ELISA. Finally, there may be some lack of specificity in serological testing of patients with other spirochetal (e.g. Leptospira or Treponema) or other borrelial diseases (e.g. Borrelia hermseii). As a result, syphilitic patients, using current diagnostic tests, may test falsely positive for Lyme Disease, and Lyme Disease patients may test falsely positive for syphilis. This is problematic due to the different treatments involved for each of these diseases as well as the social stigma attached to syphilis as a sexually transmitted disease.
Thus there remains a need for a diagnostic test which reliably detects B. burgdorferi infection in all individuals early in the course of the disease.
There are many other diseases for which a diagnostic test for use in the early stages of the disease is desirable or necessary, because the disease becomes difficult to treat, or fatal, before direct evidence of the identity of the pathogen can be found, or before the patient seroconverts.
For example, acute bacterial meningitis, particularly meningococcal meningitis (caused by Neisseria meningitidis), can be lethal within hours, with the patient rapidly progressing from symptoms such as sore throat, fever, headache, stiff neck and vomiting to drowsiness, stupor, coma and death. The use of antibiotics has reduced the fatality rate of acute bacterial meningitis to less than 10% in cases recognized early, but when diagnosed late it is often fatal. Current methods of diagnosis include a lumbar puncture followed by culturing and examination of the cerebrospinal fluid (CSF) for the presence of bacteria. However, such a test is time consuming, and lumbar puncture can cause neurological damage in the presence of a brain abscess or other mass lesion. In addition, if no bacteria are found in the culture it is difficult to determine whether viral meningitis or bacterial meningitis is the diagnosis, and therefore how to treat the patient. Due to the rapid progress of the disease, when bacterial meningitis is seriously suspected, administration of antibiotics must begin before the results of such diagnostic tests.
Similarly, septicemia (invasion of the circulation by pathogenic bacteria and their toxins) can result in septic shock, characterized by acute circulatory failure and multiple organ failure including the kidneys, lungs and heart. Mortality ranges from 25-90%, and is higher when treatment is not begun soon enough. Therefore, administration of antibiotics should begin before the results of blood cultures are known. This means treatment must be selected based on the physician's educated guess, and unnecessary agents may be administered. Further, culture results may be negative, especially in patients who have had prior antibiotic therapy, but a negative culture does not exclude septicemia.
Gonorrhea, a sexually transmitted infection caused by the bacterium Neisseria gonorrhoeae, can lead to complications such as bacteremia and gonococcal arthritis if left untreated. However, it is a disease for which there is no rapid nonculture diagnostic test. A urethral Gram stain allows identification of the causative organism in about 90% of men, but a cervical Gram stain is only about 60% sensitive in women. Nor is there a reliable serologic diagnostic test for gonorrhea currently available.
Syphilis is caused by the spirochete bacterium Treponema pallidum. There are currently two types of serological tests used to diagnose this sexually transmitted disease: non-specific screening tests which detect a substance called syphilitic reagin, and specific tests which detect antitreponemal antibodies. The screening tests are easy to perform and inexpensive, but have a high rate of false positives and do not become positive until three to six weeks after the initial infection. The treponemal tests are more accurate, but do not become positive until three to four weeks after infection. An immediate diagnosis of syphilis can be made by demonstrating T. pallidum in fluid from lesions by darkfield microscopy, but skill is needed to collect and correctly identify the organism.